Molecular phenotypes of colorectal cancer and ...

Gastroenterology Report Advance Access published September 3, 2015 Gastroenterology Report, 2015, 1–8 doi: 10.1093/gastro/gov046 Review

REVIEW

Molecular phenotypes of colorectal cancer and potential clinical applications Jonathan M. Kocarnik1,2, Stacey Shiovitz3,4, and Amanda I. Phipps1,2,* Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 2Epidemiology Department, University of Washington, Seattle, WA, USA, 3Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA and 4Department of Medicine, Division of Medical Oncology, University of Washington, Seattle, WA, USA *Corresponding author. 1959 NE Pacific Street, Box 357236, Seattle, WA 98199, USA. Tel: þ1-206-667-7741; Fax: þ1-206-667-7850; Email: [email protected] The authors wish it to be known that, in their opinion, JMK and SS should be regarded as joint first authors.

Abstract Colorectal cancer (CRC) is a heterogeneous disease, arising from many possible etiological pathways. This heterogeneity can have important implications for CRC prognosis and clinical management. Epidemiological studies of CRC risk and prognosis—as well as clinical trials for the treatment of CRC—must therefore be sensitive to the molecular phenotype of colorectal tumors in patients under study. In this review, we describe four tumor markers that have been widely studied as reflections of CRC heterogeneity: (i) microsatellite instability (MSI) or DNA mismatch repair (MMR) deficiency, (ii) the CpG island methylator phenotype (CIMP), and somatic mutations in (iii) BRAF and (iv) KRAS. These tumor markers have been used to better characterize CRC epidemiology and, increasingly, may be used to guide clinical decision-making. Going beyond these traditional tumor markers, we also briefly review some more novel markers likely to be of clinical significance. Lastly, recognizing that none of these individual tumor markers are isolated attributes but, rather, a reflection of broader tumor phenotypes, we review some of the hypothesized etiological pathways of CRC development and their associated clinical differences. Key words: colorectal cancer; microsatellite instability; CpG island methylator phenotype; KRAS; BRAF; treatment; epidemiology

INTRODUCTION As with most forms of cancer, colorectal cancer (CRC) is a biologically and epidemiologically heterogeneous disease. Such heterogeneity reflects the fact that there are many possible etiological pathways responsible for driving CRC development, each of which may be marked by distinct driver mutations and genetic or epigenetic signatures. Importantly, this heterogeneity can also have implications for CRC prognosis and the clinical management of this disease. Efforts to characterize molecular phenotypes and subtype classifications for CRC thus hold

relevance across the spectrum of the disease’s natural history— from understanding how CRC develops and who is at risk, to guiding treatment decisions and secondary prevention in an informed manner. Until recently, studies accounting for possible heterogeneity in the epidemiology and etiology of CRC have been limited to the consideration of higher-level tumor attributes, such as tumor site (e.g. colon or rectum). For example, previous studies have suggested that certain lifestyle factors, such as cigarette smoking, are more strongly associated with risk of rectal cancer

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Traditional Tumor Markers in Relation to Etiology, Epidemiology and Treatment Microsatellite instability or mismatch repair Microsatellite instability (MSI) is recognized by the presence of a high frequency of genetic alterations in microsatellite DNA repeat sequences (i.e., increased or decreased numbers of repeats in tumor DNA relative to DNA from normal surrounding tissue), resulting from deactivation of the DNA mismatch repair system. Approximately 15% of colorectal tumors exhibit high levels of MSI (MSI-high) [12]. In the majority of such tumors, MSI is due to epigenetic silencing of a DNA mismatch repair (MMR) gene (e.g. hypermethylation of the MLH1 promoter), although 20% of MSI-high tumors are due to germline mutations in one of the

MMR genes (MLH1, MSH2, MSH6 or PMS2) (i.e. Lynch Syndrome) [13]. Compared with patients with microsatellite stable (MSS) CRC, patients with MSI-high CRC are more likely to be smokers [14–17], more likely to consume alcohol [15, 16], and are less likely to be obese [18]. MSI status is consistently associated with survival of CRC: a recent meta-analysis showed MSI-high CRC to be associated with a 40% better overall survival rate than MSS CRC [12]. Even when matched for stage, individuals with MSI-high CRC—particularly in the proximal colon—appear to have a better prognosis than those with MSS CRC [19]. Emerging data suggest that therapy should be tailored to MSI status in both early-stage and advanced CRC. In particular, MSI has been shown to predict lack of benefit from adjuvant 5-FU in Stage II–III colon cancer (and possible harm in Stage II patients) [20]; however, the value of an MSI as a predictive marker with modern combination chemotherapy regimens—such as FOLFOX and FOLFIRI (5-FU with irinotecan)—is uncertain. In a recent trial of Stage III colon cancer treated with adjuvant FOLFOX, MSI status was not predictive of outcomes overall [21]. In retrospective analyses of another adjuvant trial for Stage II–III colon cancer, those with MSI-high tumors again had a better prognosis but there was no association between MSI status and benefit from oxaliplatin [22]. In contrast, MSI-high status has been shown to predict benefit from adjuvant irinotecan (IFL regimen) in at least one trial; however, MSI has not reliably served as a predictor of benefit from combination chemotherapy with 5-FU and irinotecan [23–25]. Observed differences between MSI as marker for 5-FU response vs. response to combination chemotherapy may reflect differences across trials in specific chemotherapy regimens and/or variability in multivariate models, as newer models often account for other tumor attributes, such as KRAS and BRAF mutation status [26]; thus, while MSI status is largely accepted as a prognostic marker, its role as a predictive biomarker with modern combination chemotherapy regimens remains controversial.

CpG island methylator phenotype The genome is interspersed with dense CpG-rich regions, termed ‘CpG islands’, which are found in the promoter regions of roughly half of all genes [27]. Methylation of these islands often results in gene silencing [27–29], which can be a driver of carcinogenesis (e.g. hypermethylation of the MLH1 promoter region) [30–32]. Two patterns of CpG island methylation have been observed in CRC: low-level methylation that increases incrementally with age, and high-level methylation of a particular subset of CpG islands, resulting in gene silencing [30]. CRC tumors exhibiting the latter pattern are referred to as ‘CpG island methylator phenotype (CIMP)-positive’. Approximately 30–40% of tumors located in the proximal colon and 5–15% of tumors in the distal colon and rectum can be classified as CIMP-positive [33]. Compared with CRC patients with non-CIMP tumors, those with CIMP-positive CRC are more likely to be smokers [14, 34] and less likely to be obese [35]. Although CIMP is thought to play an important role in the natural history of CRC, studies of the association between CIMP status and survival of CRC have been inconsistent [36, 37]. In part, investigation of CIMP as a prognostic or predictive biomarker has been slowed by the lack of consensus regarding which CIMP panel to use in assaying this attribute. Despite the lack of a ‘gold-standard’ CIMP assay, there has been some suggestion of an association between CIMP and a favorable response to 5-FU [38–41]; however, studies assessing the use of


than with risk of colon cancer [1, 2]; however, in light of increasing evidence indicating that the molecular profile of CRC differs greatly according to tumor site [3, 4], more sensitive epidemiological studies exploring possible etiological differences across specific molecular phenotypes of disease need to be conducted. Similarly, with respect to clinical management, the use of surgery, chemotherapy and/or radiation therapy for CRC has long been guided by the TNM stage classification and tumor site [5], without consideration of molecular attributes. Stage I (T1–2 N0) colon or rectal cancer is treated with surgery or endoscopic removal of the tumor alone. Patients with Stage II–III (T3–4 N0, Tx N1–2) rectal cancer receive, as standard, neoadjuvant chemoradiation with either 5-fluorouracil (5-FU) or oral capecitabine [6, 7]. The current standard of care for Stage III (Tx N1–2) colon cancer is adjuvant therapy, i.e. six months of 5-fluorouracil (5-FU) and oxaliplatin (FOLFOX) chemotherapy [8]. Although there is a clear benefit from adjuvant treatment in the setting of Stage III colon cancer, approximately one Stage III patient in three will still experience recurrence within five years [9]; the utility of adjuvant chemotherapy in Stage II (T3–4 N0) colon cancer remains controversial, even when it is restricted to patients with high-risk clinical features [10, 11]. The mainstay treatment for Stage IV (Tx Nx M1) colon and rectal cancer is chemotherapy; however, the poor prognosis of Stage IV CRC calls for the development of more targeted treatments; thus, biomarkers are greatly needed to tailor adjuvant therapy and more accurately guide the selection of chemotherapy regimens in CRC patients of all stages. In this review, we describe four ‘traditional’ tumor markers that have been widely studied as reflections of CRC heterogeneity: microsatellite instability (MSI) or DNA mismatch repair (MMR) deficiency, the CpG island methylator phenotype (CIMP), somatic mutations in BRAF, and somatic mutations in KRAS. The former two attributes (MSI/MMR and CIMP) represent global phenomena across the colorectal tumor genome indicative of genetic dysfunction, whereas the latter two (BRAF and KRAS mutation status) are point mutations that may act as drivers of CRC development. Here, we briefly review ways in which these tumor markers have been used to better characterize CRC epidemiology and may be used to guide clinical decision-making. Going beyond these traditional tumor markers, we also briefly review some more novel markers that are likely to be of clinical significance. Lastly, recognizing that none of these individual tumor markers are isolated attributes but, rather, a reflection of broader tumor phenotypes, we review some of the hypothesized etiological pathways of CRC development and their associated clinical differences.

Molecular phenotypes of colorectal cancer

CIMP as a predictor for response to FOLFOX therapy have not observed CIMP to be a valuable biomarker [42]; thus, the use of CIMP status as a predictive marker for conventional chemotherapy will require further investigation.

Somatic mutations in BRAF and KRAS

Additional Tumor Markers with Potential Relevance to Clinical Management

of cancer [74]. Somatic PIK3CA mutations have been noted in approximately 10–20% of CRCs [73, 75–83]. Studies characterizing patients with PIK3CA-mutated CRC have suggested that tumors exhibiting these mutations are more likely to be located in the proximal colon [77, 80, 84], and to exhibit KRAS mutations [75–80, 84] than PIK3CA-wildtype colorectal tumors. PI3K is distinct from the RAS/RAF pathway but mutations in PI3KCA may also affect responsiveness to anti-EGFR therapy, especially mutations in exon 20 [61]. PI3K pathway inhibitors have been developed, but have largely failed to show benefit in treatment [85, 86]. PIK3CA mutations may, however, suggest benefit from aspirin for secondary prevention, since large studies have demonstrated that aspirin reduces adenoma and CRC formation in individuals with PIK3CA-mutated primary CRC [87, 88].

Hypermutation Recent findings from The Cancer Genome Atlas (TCGA) network’s genome-wide analysis of CRC indicated that 16% of colorectal tumors displayed a significantly higher density of somatic sequence mutations than expected (i.e. hypermutated) [89]. The majority of these cases were also MSI-high and/or CIMP-positive, although a previously unrecognized class of hypermutated CRCs was also observed. Further investigation has led to the identification of the polymerase genes POLE and POLD1 which, when mutated in the germline or somatically, can result in this hypermutated phenotype with >1 000 000 base substitutions per tumor [90]. The clinical relevance of mutations in POLE and POLD1 and, more generally, hypermutated status, is emerging; for example, recently published data notes that MSI-high CRCs respond to programmed-death-1 (PD-1) checkpoint inhibitors, while MSS CRCs do not [91]. This finding is believed to reflect the fact that MSI CRCs have a mutation rate that is 20 times higher than that in MSS CRC, and that the neo-antigens resulting from this allow for better efficacy for immune-modulating agents. Further research into how hypermutated CRC responds to available therapies is needed, and the development of therapies targeting hypermutated CRC genomes is warranted.

Other emerging tumor markers There continues to be considerable interest in identifying predictive molecular markers for chemotherapy effect, both in the CRC adjuvant and metastatic settings [92]. Candidate biomarkers that have been heavily scrutinized include mutant TP53, thymidylate synthase (TS) expression, MEK, and amplified ERCC1, among others [93–96]. Unfortunately, none of these markers has been established as a predictive marker that is ready to be used clinically. Other future directions include dual pathway blockade to mediate mechanisms of resistance [97, 98]; however, such an approach has thus far been found to be suboptimal when applied to the clinical setting, often increasing toxicity but not improving treatment outcomes [99, 100].

Somatic mutations in PIK3CA

Molecular Classifications of CRC Subtypes Based on Proposed Etiological Pathways

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase, critical in the initiation of signaling pathways for cell proliferation, migration and survival [72, 73]. Mutations in the gene encoding the catalytic sub-unit of PI3K (i.e. the phosphatidylinositol-4, 5bisphosphate 3-kinase, catalytic sub-unit alpha [PIK3CA] gene) can result in constitutive activation of PI3K signaling and, thus, disregulated cell proliferation contributing to the development

Although much research has been devoted to the epidemiological and clinical implications of the previously-described CRC tumor markers individually, these highly correlated markers may be of even greater utility to clinical research when considered in combination. Previously-described pathways of CRC development have been suggested to result in tumor subtypes that can be distinguished by specific combinations of MSI,


Activating mutations in BRAF and KRAS are evident in approximately 5–15% and 30–45% of colorectal tumors, respectively [36, 43–48]. Such mutations result in cell proliferation and inhibition of apoptosis due to dysregulation of the MAPK signaling pathway. As part of the same pathway, BRAF and KRAS mutations tend to be mutually exclusive molecular events in CRC development [49]. The BRAF V600E mutation accounts for approximately 90% of all BRAF mutations observed in CRC [50, 51]. In comparison, mutations in ‘hot-spot’ codons 12 and 13 of exon 2 account for approximately 90% of all KRAS mutations in CRC [45, 52–54]. Compared with patients with BRAF-wildtype tumors, patients with BRAF-mutated tumors tend to be diagnosed at a later age [43], are more likely to be female, and are more likely to be smokers [14, 34]. In contrast, few epidemiological differences have been noted by KRAS mutation status [55–57]. While there is substantial evidence, from retrospective analyses of cohort and randomized clinical trials, that mutated BRAF in CRC is a marker of poor prognosis [26, 58], it is an active question as to whether BRAF-mutant patients should receive more aggressive ‘up-front’ chemotherapy. One small Phase II trial recently suggested improved survival associated with the use of FOLFOXIRI plus bevacizumab in patients with metastatic BRAF-mutated CRC [59]; however, the predictive benefit of BRAF has not been shown for either cytotoxic chemotherapy or antiepidermal growth factor receptor (EGFR) treatment [60, 61]. Notably, the success of BRAF-inhibitors seen in the treatment of melanoma has not been paralleled in CRC [62]; thus, BRAF is best considered as only a prognostic marker at this time. In contrast, KRAS mutation status is well established as a predictive marker for anti-epidermal growth factor receptor (EGFR) therapy in metastatic CRC. While initial studies of antiEGFR therapy in metastatic CRC produced mixed results, it was soon shown that efficacy of treatment could be predicted by KRAS mutation status [63–65]. Specifically, responses were observed in patients whose tumors did not exhibit KRAS exon 2 mutations, while no response—or even harm—was seen in patients with KRAS-mutant tumors [66, 67]. Multiple retrospective studies have now shown that KRAS testing restricted to exon 2 misses 15–17% of cases resistant to anti-EGFR therapy [68, 69]. ‘Expanded RAS’ testing is now advocated, which also tests exons 3 (codon 61) and 4 (codons 117 and 146) of KRAS and exons 2–4 of NRAS [70]. The utility of KRAS mutation testing for guiding treatment selection in patients with earlier stage colon cancer has not been supported [71].

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Serrated pathway

Alternative pathway

An estimated 20–30% of all CRCs develop through a serrated neoplasia pathway, named for the saw-toothed pattern of crypts in the precursor polyps [104, 107]. Precursor lesions in this pathway differ from those reflective of the traditional adenoma–carcinoma sequence, not only in appearance, but also in molecular attributes and in rates and risk of progression [104, 107–109]. Classification schemes for delineating serrated CRC based on molecular attributes continue to evolve; however, serrated CRC is generally distinguished by the presence of CIMP and mutated BRAF or KRAS [101, 105, 107]. In addition to molecular differences, several differences between serrated and non-serrated CRC have been reported in terms of genetic predisposition, anatomical site, and tumor aggressiveness [110–112]. Several studies suggest that susceptibility loci for CRC identified from genome-wide association studies (GWAS) are associated with early precursors for nonserrated CRC (adenomas), but not with serrated CRC precursors (serrated polyps) [110, 113]. Colorectal tumors exhibiting serrated molecular features are also more likely to present as proximal colon cancers than as distal colon or rectal cancers [4]. Because proximal tumors are more likely to present at later stages [114, 115], this proximal distribution of serrated CRC-defining attributes could translate to a later stage at diagnosis in serrated vs. non-serrated CRC cases; however, previous studies have not consistently demonstrated differences in the distribution of stage by BRAF mutation [37, 116, 117], CIMP [36, 118], or KRAS mutation status [37, 119]. The existence of the serrated pathway has implications for CRC screening programs: e vidence suggests that serrated tumors may develop more rapidly than other types of CRC, as the tumor markers indicative of serrated CRC are more prevalent in cancers arising within 3–5 years of a colonoscopy (i.e. interval cancers) [120, 121]. This probably reflects the more aggressive nature of CRC arising from the serrated pathway, but also probably reflects the fact that serrated polyps are often more difficult to detect using standard screening techniques: they are less likely than

Although sometimes grouped with serrated pathway cancers, colorectal tumors that are KRAS-mutated, CIMP-low, and MSS have been suggested to arise from an ‘alternative pathway’ [101, 104, 107, 123, 124]. The low levels of CIMP methylation seen in this group could represent a second type of CIMP [48], or may reflect a high level of methylation at different loci than those measured on current CIMP panels [104]. The finding that silencing of the DNA repair gene MGMT by promoter hypermethylation is associated with KRAS-mutated and CIMP-low status [124–126] suggest that MGMT methylation may be another characteristic of this alternative pathway. It is unclear which precursor lesions might be indicative of this pathway, although possibilities include traditional serrated adenomas, tubulovillous adenomas with serrated features, and serrated polyps with dysplasia [101, 107]. Although previous studies have not consistently found KRAS mutation and CIMP status individually to be significant indicators of prognosis, at least one recent study has found that CRC with a KRAS-mutated/CIMP-low phenotype indicative of this alternative pathway confer a significantly poorer prognosis than tumors derived from the traditional adenoma– carcinoma sequence [105, 106].

Traditional adenoma–carcinoma sequence

Conclusions and Future Directions With the exception of KRAS mutation testing for Stage IV CRC, current clinical practice for the management of CRC does not involve an assessment of a tumor’s molecular phenotype; however, recognizing that CRC is a heterogeneous disease, there are great opportunities to improve CRC prognosis by better incorporating information on tumor biology into treatment decisions and the design of targeted treatment strategies. Even in the absence of agents specifically targeting the treatment of CIMP-positive or BRAF-mutated or MSI-high CRC, these markers—alone and particularly in combination—provide insights into the natural history of CRC. In some instances, these markers may also serve as prognostic or predictive markers,


The majority (55–70%) of colorectal tumors arise via the wellcharacterized sequential transition from normal mucosa to adenoma to carcinoma [101–103]. This traditional pathway involves an accumulation of activating mutations in oncogenes and deactivating mutations in tumor-suppressor genes, and appears to result in CRC resulting from MSS, non-CIMP, and absent BRAF or KRAS somatic mutations [101]. Tumors resulting from this pathway are typically also characterized by driver mutations in APC and by chromosomal instability (i.e. large genomic alterations including the gain or loss of chromosomal regions and/or aneuploidy) [101, 104]. Given that this pathway is, by far, the predominating pathway responsible for CRC development, studies that have not incorporated information on CRC molecular phenotype are likely to most closely reflect the epidemiology and clinical course of tumors resulting from this pathway. In particular, these tumors tend to be associated with a more favorable prognosis than BRAF-mutated CRC, but a less favorable prognosis than MSI-high CRC [105, 106].

adenomas to bleed, making detection by fecal occult blood testing unlikely [122]; since serrated polyps are more likely to develop in the proximal colon [107], they are less likely to be identified through sigmoidoscopy and, because of their sessile, minimally elevated morphology, serrated polyps can also be difficult to detect via colonoscopy [104]. If colonoscopy and other screening methods are less efficacious for the prevention and early detection of serrated CRC than for other forms of CRC, this shortcoming will present a considerable public health challenge. In a recent analysis of CRC subtype-specific survival, colorectal tumors exhibiting a serrated phenotype marked by mutated BRAF, CIMP-positive, and MSS status, were associated with the poorest prognosis [105]; specifically, patients with these serrated cancers were more than twice as likely to die from their disease than patients with tumors exhibiting a phenotype indicative of the traditional adenoma–carcinoma sequence. In contrast however, patients with CRC exhibiting mutated BRAF, CIMP-positive, and MSI-high were significantly less likely to die from their disease than those with traditional adenoma–carcinoma pathway tumors [105]; thus, even among patients with CRC suggestive of serrated pathway origins, there is considerable heterogeneity in clinical outlook. This supports the need to consider multi-marker tumor phenotypes in projecting CRC prognosis and in guiding clinical management of disease.

CIMP, BRAF-mutation, and KRAS-mutation status. Preliminary research suggests marked differences in prognosis across these pathway-informed tumor subtypes, suggesting opportunities for more closely targeted clinical management of CRC.


Conflict of interest statement: none declared.

References 1.

2.

3. 4.

5.

6.

7.

8.

9.

Tsoi KK, Pau CY, Wu WK et al. Cigarette smoking and the risk of colorectal cancer: a meta-analysis of prospective cohort studies. Clin Gastroenterol Hepatol 2009;7:682–8, e1–e5. Cheng J, Chen Y, Wang X et al. Meta-analysis of prospective cohort studies of cigarette smoking and the incidence of colon and rectal cancers. Eur J Cancer Prev 2015;24:6–15. Yamauchi M, Lochhead P, Morikawa T et al. Colorectal cancer: a tale of two sides or a continuum? Gut 2012;61:794–97. Yamauchi M, Morikawa T, Kuchiba A et al. Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal vs. distal colorectum. Gut 2012;61:847–54. National Comprehensive Cancer Network. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines(r)) for Colon Cancer (version 20). 2015. Sauer R, Liersch T, Merkel S et al. Preoperative vs. postoperative chemoradiotherapy for locally advanced rectal cancer: results of the German CAO/ARO/AIO-94 randomized phase III trial after a median follow-up of 11 years. J Clin Oncol 2012;30:1926–33. O’Connell MJ, Colangelo LH, Beart RW et al. Capecitabine and oxaliplatin in the preoperative multimodality treatment of rectal cancer: surgical end points from National Surgical Adjuvant Breast and Bowel Project trial R-04. J Clin Oncol 2014;32:1927–34. Andre T, Boni C, Navarro M et al. Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial. J Clin Oncol 2009;27:3109–16. Hari DM, Leung AM, Lee JH et al. AJCC Cancer Staging Manual 7th edition criteria for colon cancer: do the complex modifications improve prognostic assessment? J Am Coll Surg 2013;217:181–90.

10. Tournigand C, Andre T, Bonnetain F et al. Adjuvant therapy with fluorouracil and oxaliplatin in stage II and elderly patients (between ages 70 and 75 years) with colon cancer: subgroup analyses of the Multicenter International Study of Oxaliplatin, Fluorouracil, and Leucovorin in the Adjuvant Treatment of Colon Cancer trial. J Clin Oncol 2012;30:3353–60. 11. Yothers G, O’Connell MJ, Allegra CJ et al. Oxaliplatin as adjuvant therapy for colon cancer: updated results of NSABP C07 trial, including survival and subset analyses. J Clin Oncol 2011;29:3768–74. 12. Guastadisegni C, Colafranceschi M, Ottini L et al. Microsatellite instability as a marker of prognosis and response to therapy: A meta-analysis of colorectal cancer survival data. Eur J Cancer 2010;46:2788–98. 13. Lynch HT and de la Chapelle A. Hereditary colorectal cancer. N Engl J Med 2003;348:919–32. 14. Limsui D, Vierkant RA, Tillmans LS et al. Cigarette smoking and colorectal cancer risk by molecularly defined subtypes. J Natl Cancer Inst 2010;102:1012–22. 15. Poynter JN, Haile RW, Siegmund KD et al. Associations between smoking, alcohol consumption, and colorectal cancer, overall and by tumor microsatellite instability status. Cancer Epidemiol Biomarkers Prev 2009;18:2745–50. 16. Slattery ML, Curtin K, Anderson K et al. Associations between cigarette smoking, lifestyle factors, and microsatellite instability in colon tumors. J Natl Cancer Inst 2000;92:1831–36. 17. Chia VM, Newcomb PA, Bigler J et al. Risk of microsatelliteunstable colorectal cancer is associated jointly with smoking and nonsteroidal anti-inflammatory drug use. Cancer Res 2006;66:6877–83. 18. Campbell PT, Jacobs ET, Ulrich CM et al. Case-control study of overweight, obesity, and colorectal cancer risk, overall and by tumor microsatellite instability status. J Natl Cancer Inst 2010;102:391–400. 19. Laghi L and Malesci A. Microsatellite instability and therapeutic consequences in colorectal cancer. Dig Dis 2012;30:304–9. 20. Sargent DJ, Marsoni S, Monges G et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol 2010;28:3219–26. 21. Sinicrope FA, Mahoney MR, Smyrk TC et al. Prognostic impact of deficient DNA mismatch repair in patients with stage III colon cancer from a randomized trial of FOLFOX-based adjuvant chemotherapy. J Clin Oncol 2013;31:3664–72. 22. Gavin PG, Colangelo LH, Fumagalli D et al. Mutation profiling and microsatellite instability in stage II and III colon cancer: an assessment of their prognostic and oxaliplatin predictive value. Clin Cancer Res 2012;18:6531–41. 23. Bertagnolli MM, Niedzwiecki D, Compton CC et al. Microsatellite instability predicts improved response to adjuvant therapy with irinotecan, fluorouracil, and leucovorin in stage III colon cancer: Cancer and Leukemia Group B Protocol 89803. J Clin Oncol 2009;27:1814–21. 24. Van Cutsem E, Labianca R, Bodoky G et al. Randomized phase III trial comparing biweekly infusional fluorouracil/ leucovorin alone or with irinotecan in the adjuvant treatment of stage III colon cancer: PETACC-3. J Clin Oncol 2009;27:3117–25. 25. Fallik D, Borrini F, Boige V et al. Microsatellite instability is a predictive factor of the tumor response to irinotecan in


providing even greater incentive to collect this information in clinical settings. As we evolve a better understanding of the diverse pathways that lead to CRC and improve our recognition of the driver mutations and molecular events that contribute to those pathways, approaches to the clinical management of CRC will also need to evolve and improve. Aggressive serrated BRAF-mutated/CIMPpositive/MSS tumors will probably necessitate more aggressive treatment and, potentially, different treatment agents than CRC exhibiting high levels of MSI or CRC resulting from the traditional adenoma–carcinoma sequence. As we continue to gain insight into the heterogeneity of CRC biology, etiology, epidemiology and clinical profile, the clinical management of CRC will continue to evolve in order to incorporate this information into clinical decision-making, to personalize and improve the care of CRC patients. Funding: This work was supported by the National Cancer Institute, National Institutes of Health (K07CA172298 to A.I.P., T32CA09168 to J.M.K.), the National Center for Advancing Translational Health Sciences, National Institutes of Health (KL2TR000421 to J.M.K.), and a Young Investigator Award from the Conquer Cancer Foundation of the American Society for Clinical Oncology (to S.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

| 5

6

26.

27. 28. 29. 30.

31.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.


patients with advanced colorectal cancer. Cancer Res 2003;63:5738–44. Roth AD, Tejpar S, Delorenzi M et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 2010;28:466–74. Bird AP. CpG-rich islands and the function of DNA methylation. Nature 1986;321:209–13. Gardiner-Garden M and Frommer M. CpG islands in vertebrate genomes. J Mol Biol 1987;196:261–82. Issa JP. CpG island methylator phenotype in cancer. Nat Rev Cancer 2004;4:988–93. Toyota M, Ahuja N, Ohe-Toyota M et al. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA 1999;96:8681–86. Kane MF, Loda M, Gaida GM et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 1997;57:808–11. Cunningham JM, Christensen ER, Tester DJ et al. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 1998; 58:3455–60. Hughes LA, Khalid-de Bakker CA, Smits KM et al. The CpG island methylator phenotype in colorectal cancer: progress and problems. Biochim Biophys Acta 2012;1825:77–85, Samowitz WS, Albertsen H, Sweeney C et al. Association of smoking, CpG island methylator phenotype, and V600E BRAF mutations in colon cancer. J Natl Cancer Inst 2006; 98:1731–38. Slattery ML, Curtin K, Sweeney C et al. Diet and lifestyle factor associations with CpG island methylator phenotype and BRAF mutations in colon cancer. Int J Cancer 2007; 120:656–63. Ogino S, Nosho K, Kirkner GJ et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 2009;58:90–96. Lee S, Cho NY, Choi M et al. Clinicopathological features of CpG island methylator phenotype-positive colorectal cancer and its adverse prognosis in relation to KRAS/BRAF mutation. Pathol Int 2008;58:104–13. Juo YY, Johnston FM, Zhang DY et al. Prognostic value of CpG island methylator phenotype among colorectal cancer patients: a systematic review and meta-analysis. Ann Oncol 2014;25:2314–27. Shiovitz S, Bertagnolli MM, Renfro LA et al. CpG island methylator phenotype is associated with response to adjuvant irinotecan-based therapy for stage III colon cancer. Gastroenterology 2014;147:637–45. Van Rijnsoever M, Elsaleh H, Joseph D et al. CpG island methylator phenotype is an independent predictor of survival benefit from 5-fluorouracil in stage III colorectal cancer. Clin Cancer Res 2003;9:2898–903. Min BH, Bae JM, Lee EJ et al. The CpG island methylator phenotype may confer a survival benefit in patients with stage II or III colorectal carcinomas receiving fluoropyrimidinebased adjuvant chemotherapy. BMC Cancer 2011;11:344. Han SW, Lee HJ, Bae JM et al. Methylation and microsatellite status and recurrence following adjuvant FOLFOX in colorectal cancer. Int J Cancer 2013;132:2209–16. Phipps AI, Buchanan DD, Makar KW et al. BRAF mutation status and survival after colorectal cancer diagnosis according to patient and tumor characteristics. Cancer Epidemiol Biomarkers Prev 2012;21:1792–98.

44. Phipps AI, Buchanan DD, Makar KW et al. KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers. Br J Cancer 2013;108:1757–64. 45. Ogino S, Meyerhardt JA, Irahara N et al. KRAS mutation in stage III colon cancer and clinical outcome following intergroup trial CALGB 89803. Clin Cancer Res 2009;15:7322–29. 46. Samowitz WS, Sweeney C, Herrick J et al. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res 2005;65:6063–69. 47. Berg M, Danielsen SA, Ahlquist T et al. DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAFPIK3CA-PTEN-TP53 related to age at disease onset. PLoS One 2010;5:e13978. 48. Shen L, Toyota M, Kondo Y et al. Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer. Proc Natl Acad Sci USA 2007;104:18654–59. 49. Rajagopalan H, Bardelli A, Lengauer C et al. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934. 50. Ikenoue T, Hikiba Y, Kanai F et al. Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors. Cancer Res 2003;63:8132–37. 51. Davies H, Bignell GR, Cox C et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54. 52. Bos JL, Fearon ER, Hamilton SR et al. Prevalence of ras gene mutations in human colorectal cancers. Nature 1987; 327:293–97. 53. Rosty C, Young JP, Walsh MD et al. Colorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features. Mod Pathol 2013; 26:825–34. 54. Imamura Y, Lochhead P, Yamauchi M et al. Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review. Mol Cancer 2014; 13:135. 55. Weijenberg MP, Aardening PW, de Kok TM et al. Cigarette smoking and KRAS oncogene mutations in sporadic colorectal cancer: results from the Netherlands Cohort Study. Mutat Res 2008;652:54–64. 56. Slattery ML, Anderson K, Curtin K et al. Lifestyle factors and Ki-ras mutations in colon cancer tumors. Mutat Res 2001;483:73–81. 57. Nosho K, Irahara N, Shima K et al. Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample. PLoS One 2008;3:e3698. 58. Therkildsen C, Bergmann TK, Henrichsen-Schnack T et al. The predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic colorectal cancer: A systematic review and meta-analysis. Acta Oncol 2014;53:852–64. 59. Loupakis F, Cremolini C, Salvatore L et al. FOLFOXIRI plus bevacizumab as first-line treatment in BRAF mutant metastatic colorectal cancer. Eur J Cancer 2014;50:57–63. 60. Richman SD, Seymour MT, Chambers P et al. KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol 2009;27:5931–37. 61. De Roock W, Claes B, Bernasconi D et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory


32.

|


62.

63.

64.

65.

66.

68.

69.

70.

71.

72. 73.

74. 75.

76.

77.

78.

79.

80. Day FL, Jorissen RN, Lipton L et al. PIK3CA and PTEN gene and exon mutation-specific clinicopathological and molecular associations in colorectal cancer. Clin Cancer Res 2013;19:3285–96. 81. Abubaker J, Bavi P, Al-Harbi S et al. Clinicopathological analysis of colorectal cancers with PIK3CA mutations in Middle Eastern population. Oncogene 2008;27:3539–45. 82. Zhu YF, Yu BH, Li DL et al. PI3K expression and PIK3CA mutations are related to colorectal cancer metastases. World J Gastroenterol 2012;18:3745–51. 83. Mao C, Yang ZY, Hu XF et al. PIK3CA exon 20 mutations as a potential biomarker for resistance to anti-EGFR monoclonal antibodies in KRAS wild-type metastatic colorectal cancer: a systematic review and meta-analysis. Ann Oncol 2012; 23:1518–25. 84. Phipps AI, Makar KW, Newcomb PA. Descriptive profile of PIK3CA-mutated colorectal cancer in postmenopausal women. Int J Colorectal Dis 2013;28:1637–42. 85. Rodon J, Brana I, Siu LL et al. Phase I dose-escalation and expansion study of buparlisib (BKM120), an oral pan-Class I PI3K inhibitor, in patients with advanced solid tumors. Invest New Drugs 2014;32:670–81. 86. Ganesan P, Janku F, Naing A et al. Target-based therapeutic matching in early-phase clinical trials in patients with advanced colorectal cancer and PIK3CA mutations. Mol Cancer Ther 2013;12:2857–63. 87. Liao X, Lochhead P, Nishihara R et al. Aspirin use, tumor PIK3CA mutation, and colorectal cancer survival. N Engl J Med 2012;367:1596–606. 88. Chan AT, Ogino S, Fuchs CS. Aspirin use and survival after diagnosis of colorectal cancer. JAMA 2009;302:649–58. 89. Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012;487:330–37. 90. Briggs S and Tomlinson I. Germline and somatic polymerase epsilon and delta mutations define a new class of hypermutated colorectal and endometrial cancers. J Pathol 2013; 230:148–53. 91. Le DT, Uram JN, Wang H et al. PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med 2015;372:2509–20. 92. Shiovitz S and Grady WM. Molecular markers predictive of chemotherapy response in colorectal cancer. Curr Gastroenterol Rep 2015;17:431. 93. Huh JW, Lee JH, Kim HR. Pretreatment expression of 13 molecular markers as a predictor of tumor responses after neoadjuvant chemoradiation in rectal cancer. Ann Surg 2014;259:508–15. 94. Noda E, Maeda K, Inoue T et al. Predictive value of expression of ERCC 1 and GST-p for 5-fluorouracil/ oxaliplatin chemotherapy in advanced colorectal cancer. Hepatogastroenterology 2012;59:130–33. 95. Viguier J, Boige V, Miquel C et al. ERCC1 codon 118 polymorphism is a predictive factor for the tumor response to oxaliplatin/5-fluorouracil combination chemotherapy in patients with advanced colorectal cancer. Clin Cancer Res 2005;11:6212–17. 96. Zimmer L, Barlesi F, Martinez-Garcia M et al. Phase I expansion and pharmacodynamic study of the oral MEK inhibitor RO4987655 (CH4987655) in selected patients with advanced cancer with RAS-RAF mutations. Clin Cancer Res 2014;20:4251–61. 97. Bertotti A, Migliardi G, Galimi F et al. A molecularly annotated platform of patient-derived xenografts (“xenopatients”) identifies HER2 as an effective therapeutic


67.

metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010;11:753–62. Falchook GS, Long GV, Kurzrock R et al. Dabrafenib in patients with melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial. Lancet 2012;379:1893–901. Douillard JY, Siena S, Cassidy J et al. Final results from PRIME: randomized phase 3 study of panitumumab with FOLFOX4 for first-line treatment of metastatic colorectal cancer. Ann Oncol 2014;25:1346–55. Van Cutsem E, Kohne CH, Hitre E et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med 2009;360:1408–17. Bokemeyer C, Bondarenko I, Makhson A et al. Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol 2009;27:663–71. Lievre A, Bachet JB, Boige V et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008;26:374–79. Amado RG, Wolf M, Peeters M et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26: 1626–34. Douillard JY, Oliner KS, Siena S et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med 2013;369:1023–34. Heinemann V, von Weikersthal LF, Decker T et al. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as firstline treatment for patients with metastatic colorectal cancer (FIRE-3): a randomised, open-label, phase 3 trial. Lancet Oncol 2014;15:1065–75. National Comprehensive Cancer Network. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines(r)) for Colon Cancer V.3.2014. http://www.nccn. org. Accessed June 16, 2014. Alberts SR, Sargent DJ, Nair S et al. Effect of oxaliplatin, fluorouracil, and leucovorin with or without cetuximab on survival among patients with resected stage III colon cancer: a randomized trial. JAMA 2012;307:1383–93. Manning BD and Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007;129:1261–74. Chong ML, Loh M, Thakkar B et al. Phosphatidylinositol-3-kinase pathway aberrations in gastric and colorectal cancer: meta-analysis, co-occurrence and ethnic variation. Int J Cancer 2014;134:1232–38. Samuels Y and Velculescu VE. Oncogenic mutations of PIK3CA in human cancers. Cell Cycle 2004;3:1221–24. Nosho K, Kawasaki T, Ohnishi M et al. PIK3CA mutation in colorectal cancer: relationship with genetic and epigenetic alterations. Neoplasia 2008;10:534–41. Liao X, Morikawa T, Lochhead P et al. Prognostic role of PIK3CA mutation in colorectal cancer: cohort study and literature review. Clin Cancer Res 2012;18:2257–68. Whitehall VL, Rickman C, Bond CE et al. Oncogenic PIK3CA mutations in colorectal cancers and polyps. Int J Cancer 2012;131:813–20. Ogino S, Nosho K, Kirkner GJ et al. PIK3CA mutation is associated with poor prognosis among patients with curatively resected colon cancer. J Clin Oncol 2009;27:1477–84. Velho S, Oliveira C, Ferreira A et al. The prevalence of PIK3CA mutations in gastric and colon cancer. Eur J Cancer 2005; 41:1649–54.

| 7

8

|


113. Zhang B, Shrubsole MJ, Li G et al. Association of genetic variants for colorectal cancer differs by subtypes of polyps in the colorectum. Carcinogenesis 2012;33:2417–23. 114. Minoo P, Zlobec I, Peterson M et al. Characterization of rectal, proximal and distal colon cancers based on clinicopathological, molecular and protein profiles. Int J Oncol 2010;37:707–18. 115. Hemminki K, Santi I, Weires M et al. Tumor location and patient characteristics of colon and rectal adenocarcinomas in relation to survival and TNM classes. BMC Cancer 2010;10:688. 116. Kalady MF, Dejulius KL, Sanchez JA et al. BRAF mutations in colorectal cancer are associated with distinct clinical characteristics and worse prognosis. Dis Colon Rectum 2012;55:128–33. 117. Farina-Sarasqueta A, van Lijnschoten G, Moerland E et al. The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol 2010;21:2396–402. 118. Sanchez JA, Krumroy L, Plummer S et al. Genetic and epigenetic classifications define clinical phenotypes and determine patient outcomes in colorectal cancer. Br J Surg 2009;96:1196–204. 119. Imamura Y, Morikawa T, Liao X et al. Specific mutations in KRAS codons 12 and 13, and patient prognosis in 1075 BRAF wild-type colorectal cancers. Clin Cancer Res 2012; 18:4753–63. 120. Arain MA, Sawhney M, Sheikh S et al. CIMP status of interval colon cancers: another piece to the puzzle. Am J Gastroenterol 2010;105:1189–95. 121. Nishihara R, Wu K, Lochhead P et al. Long-term colorectalcancer incidence and mortality after lower endoscopy. N Engl J Med 2013;369:1095–105. 122. East JE, Saunders BP, Jass JR. Sporadic and syndromic hyperplastic polyps and serrated adenomas of the colon: classification, molecular genetics, natural history, and clinical management. Gastroenterol Clin North Am 2008;37:25–46. 123. Nagasaka T, Koi M, Kloor M et al. Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer. Gastroenterology 2008;134:1950–60, e1. 124. Ogino S, Kawasaki T, Kirkner GJ et al. CpG island methylator phenotype-low (CIMP-low) in colorectal cancer: possible associations with male sex and KRAS mutations. J Mol Diagn 2006;8:582–88. 125. Ogino S, Kawasaki T, Kirkner GJ et al. Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer. Gut 2007;56:1564–71. 126. Whitehall VL, Walsh MD, Young J et al. Methylation of O-6-methylguanine DNA methyltransferase characterizes a subset of colorectal cancer with low-level DNA microsatellite instability. Cancer Res 2001;61:827–30.


target in cetuximab-resistant colorectal cancer. Cancer Discov 2011;1:508–23. 98. Migliardi G, Sassi F, Torti D et al. Inhibition of MEK and PI3K/ mTOR suppresses tumor growth but does not cause tumor regression in patient-derived xenografts of RAS-mutant colorectal carcinomas. Clin Cancer Res 2012;18:2515–25. 99. Dienstmann R, Serpico D, Rodon J et al. Molecular profiling of patients with colorectal cancer and matched targeted therapy in phase I clinical trials. Mol Cancer Ther 2012;11:2062–71. 100. Shimizu T, Tolcher AW, Papadopoulos KP et al. The clinical effect of the dual-targeting strategy involving PI3K/AKT/ mTOR and RAS/MEK/ERK pathways in patients with advanced cancer. Clin Cancer Res 2012;18:2316–25. 101. Jass JR. Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology 2007;50:113–30. 102. Snover DC. Update on the serrated pathway to colorectal carcinoma. Hum Pathol 2011;42:1–10. 103. Vogelstein B, Fearon ER, Hamilton SR et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525–32. 104. Leggett B and Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis. Gastroenterology 2010;138: 2088–100. 105. Phipps AI, Limburg PJ, Baron JA et al. Association between molecular subtypes of colorectal cancer and patient survival. Gastroenterology 2015;148:77–87, e2. 106. Samadder NJ, Vierkant RA, Tillmans LS et al. Associations between colorectal cancer molecular markers and pathways with clinicopathologic features in older women. Gastroenterology 2013;145:348–56, e1–2. 107. Bettington M, Walker N, Clouston A et al. The serrated pathway to colorectal carcinoma: current concepts and challenges. Histopathology 2013;62:367–86. 108. Groff RJ, Nash R, Ahnen DJ. Significance of serrated polyps of the colon. Curr Gastroenterol Rep 2008;10:490–98. 109. Li SC and Burgart L. Histopathology of serrated adenoma, its variants, and differentiation from conventional adenomatous and hyperplastic polyps. Arch Pathol Lab Med 2007; 131:440–45. 110. Burnett-Hartman AN, Newcomb PA, Hutter CM et al. Variation in the association between colorectal cancer susceptibility loci and colorectal polyps by polyp type. Am J Epidemiol 2014;180:223–32. 111. Burnett-Hartman AN, Newcomb PA, Potter JD et al. Genomic aberrations occurring in subsets of serrated colorectal lesions but not conventional adenomas. Cancer Res 2013;73: 2863–72. 112. Burnett-Hartman AN, Passarelli MN, Adams SV et al. Differences in epidemiologic risk factors for colorectal adenomas and serrated polyps by lesion severity and anatomical site. Am J Epidemiol 2013;177:625–37.